全 文 :2016
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Vol41,No.6 March,2016
ËÌÍÎÏÐ_Ñ ÒÓÔÕ²
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[
lm
]
òúË&ËÌͲÖ×ØÙÚÛ
,
éçý²²óÎÏÐ_Ñ ÒÓ
(mitogenactivatedproteinkinase,
MAPK)
ÔÕ*Øv<Wç
。
ñ^@ò
SRA
=.4$ËÌÍá\nÑè¥
、BLAST
ì³=¹Tn9
<^N
,
7¦÷=
17
ËÌÍ
MAPK
ÔÕ
,
Wò
AaMAPK1~AaMAPK17。
²
17
MAPK
ÔÕ$
,
B
16
sB
T[D/
E]Y
9<^N
。
(Ái
RealtimePCR
ú
,
úËÌͲÖ×ØÙÚÛ
17
MAPK
ÔÕÜÝÔg
。
<Äãÿ
AaMAPK3,AaMAPK10
ÜÝÛê
,MAPK7,MAPK9,MAPK12
ÜÝbê
。
Ü{úËÌÍ$Òý²²ó
MAPK
ÔÕ*
úØv
[
nop
]
ËÌÍ
;MAPK
ÔÕ
;
ØÙÚ
;
ÜÝ
[
qr)(
] 20160120
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stuT
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£bø~XZEfij
(81130070,81325023,81473307);
£bXkªÀghij
(2012BAI29B02,2012BAI28B002)
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]^s
,Tel:(010)64011944,Email:glp01@126com
MitogenactivatedproteinkinasegenesofArtemisiaannuaand
theirexpressionanalysisunderCadmiumstress
ZHOULiangyun,LVChaogeng,KANGChuanzhi,WANGSheng,TANGJinfu,KANGLiping,GUOLanping
(StateKeyLaboratoryBreedingBaseofDaodiHerbs,NationalResourceCenterforChineseMateriaMedica,
ChinaAcademyofChineseMedicalSciences,Beijing100700,China)
[Abstract] InordertostudyArtemisiaannuaundercadmiumstress,whethertherearecorespondingMAPKgenesinvolvedintrans
ductionofthecadmiumsignal17AaMAPKgenes,namedAaMAPK1AaMAPK17repectively,werefinalyobtainedbyusingTrinity
methodfordenovoassemblyoftranscriptsfromSRAdatabaseandBLASTsearchagainstAtMAPKgenesanddetermingconserveddo
mainusingaseriesofbioinformaticstoolsThereexist16MAPKgenescontainedT[D/E]Yconserveddomainsamongtheobtained
genesTheexpressionsofthesegeneswereanalyzedbyRealtimePCRundercadmiumstressTheresultsshowedthattheexpressions
levelofAaMAPK3andAaMAPK10weredownregulatedandMAPK7,MAPK9andMAPK12wereupregulatedTheseindicatedthat
thereexistcorespondingMAPKgenesinvolvedintransductionofthecadmiumsignal
[Keywords] Artemisiaannua;MAPKgene;Cadmiumstress;expressionanalysis
doi:104268/cjcmm20160607
ÎÏÐ_Ñ ÒÓ
(mitogenactivatedpro
teinkinase,MAPK)
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d]Ñ ´ÒÓ[
üÓ
,
45ý²°Xå°$
。MAPK
^èé
àB
3
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,
ì@ò
ñ^èé/bOv<¨ÛOóä3õZ
ì
[2]。MAPKKK
éX°^èébO
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]_Ò
。
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MAPKKK
@òÕ]_
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,
ejk²ÄÅ´$Ò¯Ñ Ò
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°_Òó
[3]。
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MAPKs
hi
!
,
·ì·>=.ãÿ
MAPKs
²v<
Wçòû$ZGÖühi
。
ú>8
MAPKs
ìu°Õ3
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)¨
°Õ3
(
Ö×
、
9]
、
ª
、
zÞ
、
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HÕJï
)
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[12]。
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ArtemisiaannuaL
ò&[y°
,
Fb1oò3%Wâ$,QÍ
,
DBÈîï
、
l
¡
、
ðñö
[4]。
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Ü{
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。
nQÍOwìg=
,
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、
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。
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,
ô°ËÌÍ@ò
¡¢èé/,vØv
。Liu
ö
[7]
më
,
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ÔÕuÒ
。
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,
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MAPK3
¨
MAPK6
。
ñ<ÄÜ
{
,
x9¤²Ö×ØÙÚÛv
ROSMAPK3/MAPK6。
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W ç
,
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SIMK/SAMK/MMK2/
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Archive)
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www454com/products/analysissoftware/)
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*=
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SFF
^$¹Tv=r
FASTA
þ£¹T^
。
iá\Ñèé
Trinity
(htp://trinityrnaseqsourceforgenet)
)
5
4
FASTA
¹T^nÑè
,
Xi*=
seqTypefa
singleCPU8,
ê)u¹=.Ñèá\N¹T
。
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,¨
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PÜá\N
(Unigene)。
23 AaMAPK
üÓÔÕ÷q
ìx9¤
MAPKs
ò
query
¹T
,1×10-6
hò
EValue
åv
2
,
nNF
BLAST,
³=¹Tn9N¨
Pfam
,
ì÷qËÌÍ
MAPKs
ÔÕ
。
24
2:;
Unigene
$ê$
AaMAPKs
Ô
Õ¹T
,
î.
RealtimePCR
2:;ÿ4>
@ò
PrimerPremier50
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·7101·
2016
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3
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41
áà
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}
Vol41,No.6 March,2016
`>723å=¹ÔÕ&(ÃÄBCDE
=
。
25
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RNA
pqOæ¢vö
-80
℃
ÝZq$¥(ʸ
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$çÑ
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iYè
§Çf§Ç
。
u
RNA
pq*QIAGEN
DE
RNeasy PlantMiniKit(catnos74903and
74904)¨ RNaseFreeDNaseSet(catno79254)
ù
{yh
。
Xi
Takara
DEÒá\¥1µ
(Primer
ScriptTMⅡ 1stStrandcDNASynthesisKit)nÒá
,
°¯
cDNA。
Ýb³ØÒóõò
10μL,
às
5μLPowerSYBR GreenPCRMasterMix
(AppliedBiosystems,USA),1μLcDNA,1μLprim
ers(
bÛO2w9=
,
x2ö+ò
25
mmol·L-1),ddH2O3μL,² ABI7500vöÁ
bnÒó
。PCR
¾ò
:
Ó_
(50℃,2
min);
:¯
(95℃,10min);¯
(95℃15s),
áé
¨<ê
(60℃1min)40
NM
。
7~#×ÐÈ
:Ø
。
²vö<ĵi
2-ΔΔCT
ú;=
[9]。
26
=.I
(<ÄXi
Excel2007
n=
.`ëq
。
3
<Ä
31
=.¥
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4$=.¥
,
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25168
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1000bp
Contig
aB
3292
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500bp
Contig
aB
12743
。
¥~
Contig
Ý:±ò
627
bp,
7±
Contig
ò
5709bp,
$
N50ò691bp,N90
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357bp。
32 AaMAPKs
ÔÕ2.±qu
@ò
Blast¨
<^N
,
²ËÌÍ
Unigene
=.$=
r
17
¹T
,
$B
10
TEY
d
MAPKs
Ô
Õ
,6
TDY
d
MAPKs
ÔÕ
,1
b¶d
。
î.ìJr
MAPKs
ÔÕ¹T:;`=²ôÔ
Õ.±2
,
<ÄÜ
1。
x
MAPKs
ÔÕW
B
TEY
¨
TDY2
d
,
ñ[=r¹T±0]
W
。
(<Äãÿ³B2
PCR
Æí×Ð
È:é©
,
Ü{2.±$
。
x2í¯-
(Tm)_ò:MAPK17908;MAPK27575;
MAPK37723;MAPK4763;MAPK57463;MAPK6
7445;MAPK77556;MAPK87815;MAPK97297;
MAPK107519;MAPK117815;MAPK12789;
MAPK137878;MAPK147518;MAPK16776;
MAPK177815。
Ü
1 qRTPCR
$
AaMAPKs
ÔÕ2¹T
Table1 TheprimersofAaMAPKsusedinqRTPCR
ÔÕ 2
(5′3′)
Ʊ
/bp
d
MAPK1
bO
:AAGTTCATCCTGCTGCCATTG
ÛO
:CCTTCATCTGTTCCTCGGTTG
192 TEY
MAPK2
bO
:CATCCTCGCCAGTCATTATC
ÛO
:CTTCAACTGTGATTCGTTTGG
109 TEY
MAPK3
bO
:TGGAGATCGCTTTGAGGGA
ÛO
:CGGGGGACACATTAGGGAA
198 TEY
MAPK4
bO
:TCATGCACCGAAGAACATATC
ÛO
:CATCACCTACACACACATACC
96 TEY
MAPK5
bO
:GAGGAGATGTTGAGGGAAATG
ÛO
:CGAGCCTTGGCTATCTTAAC
82 TEY
MAPK6
bO
:GTACCCGTGGATTTGGATATAG
ÛO
:GCAACAGCTTCAGGATGATA
98 TEY
MAPK7
bO
:GGCAACTCCAGTAGAACCACC
ÛO
:GCAAACGATCCCATAGGCAC
139 TEY
MAPK8
bO
:CAAAGAGGATTAGCGTGACAGAA
ÛO
:GGCAGTAGCAAGAACTTCAGGAT
188 TEY
MAPK9
bO
:GAAAGGTGTTCCGGTGTATG
ÛO
:CGGCCAACAGGTTGAATAG
105 TEY
MAPK10
bO
:AATGCTTTTGATAACCGTATTGATG
ÛO
:GTGCTTGAGACGACTTGATAATCTG
196 TEY
MAPK11
bO
:CAAGTTCCTCAAAGCGTCCA
ÛO
:CCTTCGTTGTTCAAGAGCCG
102 TDY
MAPK12
bO
:CAACAGGAACCCATATTACAACC
ÛO
:GGAAGAAGAGCCTCAAGCAAA
196 TDY
MAPK13
bO
:ACACCCAAATATGTAGGAGCCA
ÛO
:TTGAAGCCTTGATACCCGAAT
128 TDY
MAPK14
bO
:CCATCAAGGCGGGAATTTA
ÛO
:TGCTCTTGTGTCAAGTCATC
101 TDY
MAPK15
bO
:CCTCAACGACCGTCTATCTATTCC
ÛO
:GCTATTGTTACTTCCTGATTCTATGGC
135 TDY
MAPK16
bO
:CACGGAGTATGGAGAAGCGAGT
ÛO
:CGGATGCCTAAGCAAACGAA
199 TDY
MAPK17
bO
:GGGCACCAGAACTCATTTTTG
ÛO
:AATAGTGGCTGTCCCAGAAGG
103 —
33 AaMAPKs
ÔÕÜݱ
²ËÌÍ
Unigene
$ a ì J r
17
MAPK
Ô Õ
,
$
MAPK15
²ËÌÍ{ü$ÜÝöÃH
,
Õ1²N¥
($Bqu²ÜÝö
。16
AaMAPKs
Ô
Õ²ÜÝöq
1。
7qFGIOs
<±
,AaMAPK3,AaMAPK102
ÔÕ²\$
ÜÝöl¦°
20,80μmol·L-1I,ù{
úËÌͲØæçÛ
AaMAPK3,AaMAPK10
Ô
ÕÜݪÝrú±ù
。AaMAPK3,AaMAPK10
: °
TEY
d Ô Õ
。AaMAPK7,AaMAPK9,
AaMAPK12
²
20,80μmol·L-1I$ÜÝö
:°\9IË·ÜÝö·
,
ù{
ØËÌÍ$
AaMAPK7,AaMAPK9,AaMAPK12
ÜÝÜë$Îhi
。
$
AaMAPK7,AaMAPK9
·8101·
±#ö
:
ËÌÍÎÏÐ_Ñ ÒÓÔÕ²Ö×ØÙÚÛÜÝ
°
TEY
d
,AaMAPK12
°
TDY
d
。
FG
IOs¯_
,AaMAPK6
ÔÕ²\¨xI
$ÜÝ&>ÔN[÷
,
9²°
0h
ÜÝö:
Ã
。
Ëgr²qòû$üîq{ü
,
°
ú
,
Õ1{u
AaMAPK6
ÔÕÜÝÒ
fïBô
,
ù{fï²[vûbÒ
Î
AaMAPK6
ÜÝ
。
@ò<^N
AaMAPK6
ÔÕ°
TEY
d
。
q
1 AaMAPKs
ÔÕÜݱ
Fig1 ThediferentialexpressionanalysisofAaMAPKs
4
õã
/ËÌÍ
SRA
=.4$
5
EST
4n¥
=rú´öÃËÌÍ
Unigene,
òìJËÌÍ
AaMAPKs
ÔÕp)`qJ=.4
。
íî
,
wB
ú>
MAPK
ÔÕu$ì
,
$x9¤B
20
[10],
ªðB
17
[11],
¬QB
19
[12],
ña
17
[13],
«
16
[14]。
N
@ò
Blast¨
<^N
,
²ËÌÍ
Unigene
=.4$aqJrú
17
¹T
。
$B
10
TEY
d
MAPKs
ÔÕ
,6
TDY
d
MAPKs
ÔÕ
,1
b¶d
。
ê²5
£x9¤$
20
MAPKs
ÔÕ$
,12
°
TEY
d
,8
°
TDY
d
[15]。
ù{£¤?
ðs
MAPK
ÔÕ=í¨<^Nd£¤
,
þÔ
ÕÒ*£¤°/
。
(@òØæçÛËÌÍ$
MAPK
ÔÕÜ
Ýn±
,
mëB
2
MAPK
ÔÕ
(AaMAPK3
¨
AaMAPK10)
ÜÝöÛê
,3
MAPK
ÔÕ
(MAPK7,MAPK9,MAPK12)
ÜÝöb
ê
,
ù{úËÌÍ$ý²²ó
MAPK
ÔÕ*
úØv<Wçhi
。
Bòé
,
N
$më
AaMAPK3,AaMAPK102
ÔÕ²Ö×ØhiÛ
ÜÝöÛî
。
êríîò§Bô²Ö×Ø
|,>hiÛ
,MAPK
ÔÕÜÝÛêBk
.
[16]。
¤O
,
ÜÝÛê
MAPK
ÔÕ²v
2016
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3
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41
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Vol41,No.6 March,2016
çòû$1Gÿ?hiIb¶
。
r,
,
@ò
(=.{uËÌÍ$ý²[
MAPK
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fïB¶óhi
,`
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Ýöø
。
[
yg?z
]
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°ó
,
£!õ
,
ûûû
,
ö
.
F
MAPK
ÔÕã䪺Ù
ÚÛÜÝ
[J].
52>Fk
,2015(3):4.
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